Biolog identification Bacterial contamination in kerosene storage tanks: intraspecific biodiversity and bioremediation potential of isolates Iulia CHICIUDEAN1, Ana-Maria TANASE1,2, Ioana MEREUTA1, Robertina IONESCU1, Tatiana VASSU1, Ileana STOICA1* Department of Genetics, University of Bucharest, Romania * corresponding author: Ileana Stoica: prutu@yahoo.com Molecular Biology Laboratory, Faculty of Biotechnologies, University of Agronomic Sciences and Veterinary Medicine of Bucharest INTRODUCTION MICROBIAL HYDROCARBON-DEGRADING POTENTIAL Microbial contamination of storage tanks, as well as pipes, poses serious economic problems due to biofilm formation, clogging of valves and fuel deterioration. Kerosene is a jet engine fuel with mainly a paraffinic composition. Because of its composition, kerosene hydrocarbons are generally not inhibitory to microbial activity and kerosene biodegrade significantly under aerobic conditions. The aim of this work was to investigate the biodiversity of such aerobic bacterial contaminants in kerosene storage tanks, biofilm formation capacities and alkanes degradation potential of those contaminants. A. Screening for alkane hydroxylase genes PCR with ALK-3R / ALK-3F Kohno, 2002 set of primers: 5'-TCGAGCACATCCGCGGCCACCA-3' 5'-CCGTAGTGCTCGACGTAGTT -3' 500pb 300pb 330pb Detection of alkB gene in enrichment isolates. Lane 1: 100bp ladder, 2: E1, 3: E2, 4: E3, 5: E4, 6: E5, 7: E6, 8: E7, 9: E8, 10: E9, 11: E10. ISOLATION OF BACTERIAL STRAINS B. Bacterial growth on n-alkanes Water samples were collected from the water/fuel interface from seven kerosene storage tanks; Bacteria were isolated using enrichment cultures (MSM - Minimal Salts Medium supplemented with hydrocarbons) 10 isolates were obtain from four type of enrichment cultures Enrichment cultures Isolated strains MSM media 5%(v/v) Fluka oil E1, 2, 3 5%(v/v) n-hexadecane E4, 5 5%(v/v) n-hexadecane + 5%(v/v) Fluka oil E6, 7 10% (wt/v) naftalene E8, 9, 10 Growth rate of the Pseudomonas strains isolated from enrichment cultures when incubated for 21 days on MSM supplemented with n-decane (C10), n-dodecane (C12) or n-hexadecane (C16) as soul carbon sources. n = number of generations. BIOLOG IDENTIFICATION Bacteria isolates Biolog identification Similarity index E1 Pseudomonas fluorescens biotype C 0.776 E6 Pseudomonas synxantha 0.574 E2 0.851 E7 0.588 E3 0.623 E8 Pseudomonas sp. 0.486 E4  0.753 E9 0.506 E5  0.879 E10 0.707 BIOFILM FORMATION ASSAY Cell adhesion → biofilm formation capacities → fuel systems infrastructure problems caused by microbes (microbiologically influenced corrosion (MIC) and fouling) Reading the optical density at 590nm of sessile cells → proportional to the number of attached cells in the microtiter plate wells Biofilm formation capacity for all kerosene storage tanks isolates. The height of the bars represent the biofilm formed after 24 hours (x-axis) by the pure strains. Error bars display standard deviation and refers to the total biofilm formation based on four replicate wells perplate. Phenogram retrieved from BIOLOG-software: rooted phylogenetic tree showing relationship among enrichment culture isolated strains (UPGMA algorithm). Dendrogram retrieved from Biolog-program for identification of E10 strain CONCLUSIONS Biolog MicroLog analysis identified our isolates from water samples collected from the water/fuel interface from seven kerosene storage tanks as Pseudomonas fluorescens biotype C (E1, E2,E3,E7, E10), as Pseudomonas synxantha (E4,E5,E9), and Pseudomonas sp. E8; Local phylogenetic tree generated by Biolog-software shows that even the strains are close to each other, clustering in 3 majore groups, they are not identical; ARDRA profiles sugested that all 10 isolates belong to Pseudomonas fluorescence specie, not discriminating between Pseudomonas fluorescence and Pseudomonas syxantha; For all 10 enrichment cultures isolated strains we obtained the amplicon of interest (330bp) → alkane hydroxylase AlkB gene is present in the genome of all P. florescens isolated strains; All the P. fluorescens investigated genotypes were able to use n-decane (C10) and n-dodecane (12), even more, n-decane (C10) and n-hexadecane (C16) supported a growth rates that exceeded eigth generations (when used as sole carbon source); The ability to adher to the polystyrene microplate well surface was observed for all strains, but only for E5 we register the highest adhesion capacities OD590nm>1.8; Our study showed that bacterial strains isolated from kerosene storage tanks has intraspecific biodiversity and a high bioremediation potential; MOLECULAR BASED IDENTIFICATION ARDRA (Amplified Ribosomal DNA Restriction Analysis) 16s rRNA gene amplicon (primer pair 27F/1492R) ARDRA restriction profiles (2% agarose gel) with CfoI (left) and XbaI (right). Lanes M, 100 bp DNA ladder (Promega); lanes 1–6, strains E1, E7, E6, E4, P. fluorescens ATCC 49642, P. aeruginosa ATCC 27893. Reference: 1. Kohno T., Sugimoto Y., Sei K., Mori K., (2002) Design of PCR primers and gene probes for general detection of alkane-degrading bacteria, Microbes and Environments, vol. 17, no. 3:114-121. 2. Smits T., Balada S. B., Witholt B., van Beilen J.B., (2002) Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria, J Bacteriol., vol.184, no. 6: 1733–1742. Acknowledgements: